Methods for treating breast cancer using a mammary cell growth inhibitor

ABSTRACT

Mammastatin has an approximate molecular weight of 44 kDa in its inactive, non-phosphorylated form. Normal mammary cells express functional phosphorylated forms having approximate molecular weights of 53 kDa and 49 kDa. Metastatic mammary cells either do not express Mammastatin at all, or do not express the 53 kDa or 49 kDa, phosphorylated forms. Mammary cancer cells are inhibited in their growth by the administration of phosphorylated Mammastatin.

RELATED APPLICATIONS

This application is a divisional of Ser. No. 09/369,212, filed Aug. 6,1999, issued Dec. 10, 2002 as U.S. Pat. No. 6,492,504, which is acontinuation of application Ser. No. 09/285,379, filed Apr. 2, 1999issued Sep. 17, 2002 as U.S. Pat. No. 6,451,765, which is a divisionalof 08/943,828, filed Oct. 3,1997, now abandoned, which claims thebenefit of priority to U.S. Provisional Patent Application Ser. No.60/027,315, filed Oct. 3, 1996, now abandoned, all of which are herebyincorporated in their entirety.

BACKGROUND OF THE INVENTION

Breast cancer is a disease that kills over 45,000 women each year in theUnited States alone. Over 180,000 new cases of breast cancer arediagnosed annually, and it is estimated that one in eight women willdevelop breast cancer. These numbers indicate that breast cancer is oneof the most dangerous diseases facing women today. Cancer research hasbeen unable to determine the cause of breast cancer, and has not found asuitable method of therapy or prevention.

A woman diagnosed with breast cancer may be treated with surgery,hormone therapy, chemotherapy, and radiation. If the patient developsmetastatic disease, radiation and high dose chemotherapy are required toablate the cancer in remote areas such as the brain, bone, and liver.

The current therapies available for the treatment of breast cancer aretoxic, dangerous, costly, and many are ineffective, especially in thetreatment of metastatic disease. The table below was extracted fromChurchill Livingston, Clinical Oncology, 1995, and summarizes dataavailable on the current methods of treatment and expected survivalrates.

Treatment Method Effect Toxicity Result Survival adriamycin bolus killcancer high can induce +14 months cells remission cyclophosphate boluskill cancer high can induce +16 months cells remission methotrexateinfusion kill cancer high can induce +16 months cells remission 5Furacil infusion kill cancer high can induce +18 months cells remissionmix of above mixed kill cancer high can induce +22 months cellsremission taxol bolus kill cancer high can induce +12 months cellsremission estrogen oral may stop low can induce +6 months growthremission tamoxifen oral may stop low may stop +12 months growthprogression mastectomy surgery remove tumor low may eliminate +5 years*cancer lumpectomy surgery remove tumor low may eliminate +5 years*cancer surgery and combination combination low may eliminate +7-10years* tamoxifen cancer radiation mechanical kill cancer high can induce+14 months cells remission *assumes there are no micrometasteses

Currently, there are no therapies that are effective for long termtreatment of breast cancer that has metastasized to lymph nodes ordistal sites. Local disease can be effectively treated by surgery, ifall of the cancer can be removed. A new therapy for the effectivetreatment of breast cancer that could stop the growth of breast cancerand of cells derived from metastatic cancer is urgently needed. Such atherapy would be useful in the treatment of localized breast cancer, inlong term treatment of metastatic disease, and as a follow-up treatmentafter surgical removal of tumors. Other applications include a growthinhibitor as a primary therapy and for preventative use.

Detection methods for breast cancer, such as mammogram, physical exam,CAT-scan, and ultrasound, have significantly improved early detection ofbreast cancer. However, with these methods, a suspected tumor must stillbe surgically removed for pathological examination to determine if thetumor is benign or malignant, and to attempt to determine the tissuetype and grade of the malignancy. This pathological diagnosis helps todetermine what subsequent treatment protocols may be used.

For breast cancer, these methods are generally inconclusive, as adequatebreast cancer tumor markers are not available. Available markers such asCA 15-3 and CA 27-29 are used as indicators of metastases, however, theyare not specific. There is a great need for diagnostic tools and methodsthat can effectively and reliably diagnose breast cancer, e.g., usingnew and specific breast cancer markers. In addition, a reliable andsimple method for the early detection and diagnosis of breast cancer isgreatly needed. Preferably, such an early detection method wouldidentify breast cancer in its early stages, track progression of breastcancer through advanced metastatic disease, and diagnose the propensityof a patient to develop breast cancer or to develop advanced disease.Most preferably, the diagnostic method could be used without tissuebiopsy, e.g., by analysis of a body fluid such as blood.

Human mammary tissues undergo a burst of proliferative activities at theonset of menarche and during each menstrual cycle. Studies on theeffects of estrogen on mammary tissues and tumors indicate that estrogenis a primary growth-initiating factor for mammary tissues.Estradiol-sensitive growth factors have been characterized. In addition,mammary cell growth factors which are not hormonal in nature have alsobeen described.

Specific growth factors which have been shown to have a stimulatingeffect on mammary tissue growth include platelet-derived growth factor(PDGF), insulin-like growth factor (IGF-1) and transforming growthfactor (TGF) alpha. TGF-beta, on the other hand, has been shown tosuppress mammary tissue growth.

The regulation of mammary cell growth is of great importance in thediagnosis and treatment of breast cancer. Neoplastic growth of mammarytissues, if unchecked, can develop into uncontrollably-proliferatingmalignant tumors, which are the cause of death of thousands of womenyearly. A growth inhibition factor capable of specifically suppressingmammary cell growth would provide a dynamic tool for use in thediagnosis and treatment of breast cancer.

Thus, it would be of great utility to isolate and characterize aspecific mammary cell growth inhibitor, to identify its nucleic acidsequence and amino acid sequence, and to recombinantly express theinhibitor as a purified protein. Diagnostic and therapeutic methodsusing the nucleic acid sequence and/or recombinantly produced inhibitorwould be of great utility in the diagnosis and treatment of breastcancer.

SUMMARY OF THE INVENTION

A specific mammary cell growth inhibitor, Mammastatin, has been isolatedfrom normal human mammary cells and characterized. It has now been foundthat Mammastatin is produced by normal mammary cells, but not by breastcancer cells. Furthermore, it has now been found that the reduction orabsence of Mammastatin in the blood correlate with the presence ofbreast cancer. Administration of active Mammastatin prevents growth ofbreast cancer cells.

The nucleic acid sequence encoding Mammastatin has now been cloned,sequenced, and expressed recombinantly in host cells as an activeinhibitor of mammary cell growth. The isolated and characterized nucleicacid sequence (SEQ ID NO.: 1) and its deduced amino acid sequenceprovide unique and specific tools for use in the diagnosis and treatmentof breast cancer.

The present invention provides an isolated and purified nucleic acidsequence encoding Mammastatin, a specific protein inhibitor of mammarycell growth, and particularly of mammary cancer cell growth. Theinvention also includes plasmids and vectors containing the Mammastatinnucleic acid sequence, amino acid sequence of Mammastatin, and methods,kits, and compositions utilizing the Mammastatin nucleic acid or aminoacid sequences to produce purified mammary cell growth inhibitor and inthe diagnosis and treatment of breast cancer. The inventive compositionsinclude probes and primers that specifically hybridize to theMammastatin nucleic acid sequence and its RNA products.

The invention further includes a method for treating breast cancer byadministering Mammastatin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a Western Blot showing expression of recombinant Mammastatinin Eucaryotic Cos-7 cells.

FIG. 2 is an immunoblot showing expression of Mammastatin in insectcells.

FIG. 3A is an autoradiograph showing expression of recombinantMammastatin produced by in vitro transcription and translation and usedin the growth inhibition assays of FIG. 3B. FIG. 3B is a graph showinginhibition of mammary cell growth by recombinant Mammastatin produced byin vitro transcription and translation.

FIG. 4 is a graph showing growth inhibition in human mammary cancer cellgrowth by treatment with conditioned medium of Cos-7 cells transfectedwith Mammastatin cDNA.

FIG. 5 is a Western Blot showing relative amounts of 53, 49 and 44 kDMammastatin in normal and cancerous human mammary cells.

FIG. 6 is an immunoblot showing phosphatase digestion of Mammastatin.

FIG. 7 is a graph showing the effect of phosphatase on the activity ofMammastatin.

FIG. 8 is a Western Blot showing Mammastatin from normal and canceroushuman mammary cells, as well as in mixed cultures of normal andcancerous cells.

FIG. 9 is a graph showing a Mammastatin ELISA standard curve.

FIG. 10 is a graph showing Mammastatin in normal human serum as analyzedby ELISA.

FIG. 11 is a graph showing Mammastatin levels in breast cancer patientsover the course of treatment.

FIG. 12 is a Western blot showing expression of Mammastatin induced byretrovirus.

FIGS. 13A, 13B and 13C are graphs showing the effect of Mammastatintreatment on MCF7 tumor cells in nude mice.

FIGS. 14A, 14B and 14C are graphs showing the effect of Mammastatintreatment on tumor cells in nude mice.

FIG. 15 is a dot blot assay showing Mammastatin in blood from normalfemales versus the absence of Mammastatin in blood from breast cancerpatients.

FIG. 16 is a Western blot showing recombinant Mammastatin expression inCos-7 cells.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Mammastatin

Mammastatin is a protein growth inhibitor produced and secreted bynormal human mammary epithelial cells. A mammary cell growth inhibitorwas first described as an inhibitory protein activity present in mediaconditioned by the growth of normal human mammary cells. The inhibitoryactivity was identified in conditioned medium from normal human mammarycells, but not in media conditioned by the growth of human mammarycancer cells. The inhibitory activity was determined by bioassay andantibody development to reside in three proteins, having the approximatemolecular weights of 53, 49 and 44 kD (Ervin, Paul R., DoctoralDissertation University of Michigan, 1995).

It has now been determined that a specific mammary cell growthinhibitor, Mammastatin, is expressed as a 44 kD protein which isphosphorylated increasing the molecular weight to 49 kD and 53 kD. Thenon-phosphorylated 44 kD form is not an active inhibitor, whereas thephosphorylated 49 kD and 53 kD forms inhibit growth of breast cancercells. The active 53 and/or 49 kD phosphoprotein is expressed by normalhuman mammary cells, but is not generally produced by human mammarycarcinoma cells. Some carcinoma cells make the 44 kD protein that lacksphosphorylation and is inactive.

The table below summarizes data showing expression and activity ofMammastatin in normal and cancerous cells and tissues.

44 53/49 produce are Cell Type* Number kDa** kDa** inhibitor inhibitedNormal primary 42 +/− ++++ 42/42 2/2 cultures Normal breast tissue 5 +++ Mammary Cell Line 16  0/16 12/12 Type A 11 + −  0/11 8/8 Type B 5 − −0/5 4/4 Breast tumor lysate 25 Type A 17 + − Type B 8 − − Non-mammarycell 8 0/2 0/8 lines Type A 3 + −*** 0/1 0/3 Type B 5 − −*** 0/1 0/5*Carcinoma cells in which 44 kDa Mammastatin was detected (Type A) ornot (Type B) **(−) No expression (++++) intense expression ***Two celllines, BxPc3 and A253 expressed proteins identified as 53/49 kD, butneither cell line produced inhibitory activity.

Dose response studies with human mammary carcinoma cells indicates thatcarcinoma cell growth is 50-70% inhibited with 10 ng/ml of Mammastatinand blocked completely with 25-50 ng/ml. Highly metastatic cells such asMDA-MB-435 and MDA-MB-231 required 50 ng/ml to stop growth. In vitro andin vivo clinical data experiments indicate the effect is reversible, andthat repeated administration of the inhibitor is required to arrestcarcinoma cell growth at the lower concentrations. At doses above 50ng/ml, however, Mammastatin appears to induce apoptosis, as indicated byhistology, e.g. cell necrosis.

Since Mammastatin is a natural growth inhibitor that blocks mammarycarcinoma cell growth, and since no tumors make active Mammastatin,Mammastatin replacement therapy is ideal for therapeutic treatment ofbreast cancer. The clinical data provided in the examples belowdemonstrate the effectiveness of Mammastatin replacement therapy.

The nucleic acid sequence encoding Mammastatin protein has now beenisolated, characterized, sequenced (SEQ ID NO.: 1), determined to encodeall three (53, 49, and 44 kD) molecular weight proteins, and given thename “Mammastatin”. Differences in the molecular weight of the threeforms has been determined to be caused by the extent of the protein'sphosphorylation. Mammastatin produced by normal human mammary cells(NHMC) in culture and recombinantly expressed Mammastatin inhibit thegrowth of human mammary carcinoma cells, and is useful as a therapeuticagent in the treatment of breast cancer.

Analysis of human sera from normal women and from breast cancer patientsindicates that decreased blood levels of Mammastatin correlate withadvancing breast cancer. Screening and monitoring blood serum for thepresence of this active inhibitor as described in the examples belowprovides a specific and effective diagnostic tool.

Nucleic Acid Sequence

The Mammastatin DNA nucleic acid (SEQ ID NO.:1) is shown in the tablebelow, and was identified by cloning and sequencing of Mammastatin cDNAfrom a normal human mammary cell cDNA library, as described more fullyin the Examples below. Chromatographically purified inhibitor had notpreviously been sufficiently isolated to permit its amino acid analysis,and early attempts to sequence the protein inhibitor by standardtechniques failed. Attempts to screen a cDNA library using antibodiesraised against chromatographically purified inhibitor protein failed togenerate an active clone. To overcome these problems, the gene encodingMammastatin was identified by peptide sequencing and degenerateoligonucleotide screening of a normal human mammary cell cDNA library.

Concentrated protein produced by normal human mammary cells was affinitypurified using an anti-Mammastatin antibody raised againstchromatographically purified inhibitor. Purified protein fractions weresupplemented with a small amount (10⁵ cpm) of ³²P labeled as tracers.The labeled tracer protein was purified from conditioned media of cellsgrown in the presence of ³²P, as described more fully in the examplesbelow. The protein was cleaved with cyanogen bromide, and cleavedfragments were identified as Mammastatin by autoradiographic analysis of³²P-labeled protein. The most abundant labeled peptides generated by thecleavage were sequenced.

Two peptides, selected as having unique amino acid sequences (SEQ IDNOS.: 2 and 3), were used to produce degenerate oligonucleotides. Thedegenerate oligonucleotides were then used to screen a normal humanmammary cell cDNA library.

One clone, labeled pMammA, hybridized to oligonucleotides from bothselected peptides. This clone was further characterized, and was shownto express protein recognized by anti-Mammastatin antibodies. The clonehas been verified as encoding Mammastatin by Northern blot analysis, invitro transcription and translation assays, and growth inhibitionassays. A pcDNA3 clone containing the Mammastatin cDNA insert (pMammB)was deposited with the American Type Culture Collection and givenAccession Number 97451. The recombinant protein expressed from pMammBhas been detected by immunoblot of transfected mammalian cell lines andhas been demonstrated to possess growth inhibitory activities againstmammary cancer cells. The cDNA clone has been completely sequenced (seeExample 3) and found to be unique to the BLAST DNA database.

The nucleic acid sequence of the invention (SEQ ID NO.: 1) encodes humanMammastatin, which functions to inhibit the growth of human mammarycells, normal and cancerous. The term “human” is not intended to limitthe source of the protein nor to limit its inhibitory effects only tohuman cells and tissues. It is understood that the nucleic acid sequenceand amino acid sequence of Mammastatin in individuals may vary somewhat,without altering the structure or function of the protein. Further, oneskilled in biochemistry will appreciate that modifications of thenucleic acid or amino acid sequence may be made without altering thestructure and/or function of the molecule. For example, the nucleic acidsequence may be modified to permit optimal expression of the desiredamino acid sequence using known optimal codons for a particular cellularhost.

The nucleic acid sequence of the invention is useful in producing largequantities of highly purified Mammastatin protein for use in therapeuticand diagnostic methods in the treatment of breast cancer.

Anti-Mammastatin Antibodies

Several anti-Mammastatin antibodies have been produced andcharacterized. See, for example, PCT application WO 89/11491 published+Nov. 1989. These antibodies were raised against chromatographicallypurified inhibitor protein, and have been demonstrated to block theinhibitory effect of Mammastatin protein on mammary cell growth.

Available anti-Mammastatin antibodies include 7G6 and 3C6, commerciallyavailable from Neomarkers (Freemont, Calif.) and 6B8. Hybridoma cellsproducing 7G6 are available from the American Type Culture Collection(ATCC Accession No. PTA-4606, Docket No. 10152, deposited Aug. 21, 2002,ATCC 10801 University Blvd, Manassas, Va. 20110-2209). Hybridoma cellsproducing 6B8 antibody are available from the American Type CultureCollection (ATCC No. HB 10152). Each of these antibodies binds to allthree molecular weight forms of Mammastatin and are useful inimmunological assays, including dot blots and Western blots. The 7G6antibody is preferred for Western blot analysis or for ELISA analysis ofdenatured protein samples. The antibodies 3G6 and 6B8 may be used inELISA assays, e.g., under conditions specified in the examples.

Additional antibodies can be produced using standard methods known forproducing monoclonal or polyclonal antibodies. The antigen used toproduce antibodies may be derived from culture of NHMC or fromrecombinantly expressed Mammastatin.

Diagnostic Method

The invention further provides an in vitro assay for detecting active,inhibitory Mammastatin in patient samples, including tissues, cells, andfluids. Breast cancer disease and advancing metastatic disease isdiagnosed by correlating the presence and type of Mammastatin protein ina patient's sample with that of normal or cancerous human mammary cells.A patient's blood or tissue sample is analyzed for Mammastatin protein,e.g., for the abundance of Mammastatin protein and/or for the molecularweight forms of Mammastatin. As discussed below, the absence or loss ofMammastatin, particularly of the higher molecular weight, phosphorylatedforms of Mammastatin, is correlated with breast cancer and indicative ofadvancing metastatic disease.

Analysis of Mammastatin is preferably by immunoassay, including ELISA orWestern Blot analysis of a patient's blood samples, usinganti-Mammastatin antibodies. Preferably, recombinant Mammastatinstandards are used to provide a standard curve for reliable quantitationof inhibitor levels. Such immunoassays are exemplified by the dot-blotassays and Western blot assays shown in the examples below. In analternative preferred embodiment of the invention, tissue samples, suchas tumor biopsies, are analyzed by immunohistochemistry, or by culturinga patient's tumor cells and examining the cultures for expression ofMammastatin.

In a particularly preferred embodiment, an assay for the diagnosis ofbreast cancer includes at least two specific antibodies: an antibody toidentify the sampled breast tissue as epithelial tissue, such as ananti-cytokeratin antibody, and an anti-Mammastatin antibody. Forexample, using an immunoblot format, tissue suspected of containingbreast cancer cells is homogenized, separated on an SDS/PAGE gel,transferred to membrane, and probed with both anti-keratin andanti-Mammastatin antibodies. Isotype specific second antibodies that areconjugated to a suitable marker system such as peroxidase or alkalinephosphatase are used to detect bound antibodies. Membranes containingbound first and second antibodies are then developed using knowncolorometric or fluorometric techniques and quantitated by knownmethods.

In the most preferred embodiment, the sample is analyzed for thephosphorylated forms of Mammastatin, such as by Western Blot, usinganti-Mammastatin antibodies. A decline or absence of the high molecularweight (53/49 kD) Mammastatin correlates with advancing breast cancer.

Recombinant Expression Vectors and Transformed Cells

Recombinant expression vectors of the invention are useful forproduction and amplification of purified Mammastatin protein andportions thereof, and for easy isolation of Mammastatin protein andportions thereof to be used in diagnostic and therapeutic methods.

A target sequence, such as all or a portion of the 2.4 kb MammastatincDNA (SEQ ID NO.:1), is cloned into a suitable nucleic acid sequenceexpression vector such as pUC18, pKC30, pBR322, pKK177-3, pET-3, pcDNA3(Invitrogen) for COS and CHO cells, and pAcG3X baculovirus expressionvector (PharMingen, San Diego, Calif.) for expression in insect cells,and like, known expression systems by standard methods. Commerciallyavailable expression vectors provide for cloning of a target sequenceinto a site of the vector such that the target sequence is operablylinked to transcriptional and translational control regions.

The expression vector is then introduced into suitable host cells usingknown methods such as calcium phosphate precipitation, liposome mediatedtransformation, protoplast transformation, electroporation, and thelike. Suitable host cells include COS and CHO cells, High 5 and SF9insect cells, baclovirus, and yeast cells. Other host cells include E.coli strains such as E. coli DH5α, and avirulent isogenic Salmonellaspp. such as S. typhimurium deletion mutants lacking adenylate cyclaseand cAMP receptor protein, Salmonella mutants in aro genes, and otherSalmonella vaccine strains as described in Bio/Tech, 6:693 (1988).

Preferably, the cellular host is a Eukaryotic cell, capable ofexpressing the protein with proper folding and kinase activity toproduce a phosphorylated, active inhibitor. Host cells may be screenedby transfection with cDNA encoding Mammastatin. Analysis of the proteinproduced by the transformed cells, e.g. by immunoblot, and the abilityof the protein to inhibit mammary cell growth, for example MCF7 cellgrowth, as described in the examples recited below, can be used toscreen potential host cell systems.

Host cells transformed with the target nucleic acid sequence arescreened by a variety of methods including colony hybridization orreactivity with antibodies specific for Mammastatin protein. Atransformed cell is a suitable host cell carrying a pcDNA3 or otherplasmid or vector containing a nucleic acid sequence encodingMammastatin. One such plasmid is the pcDNA plasmid (pMammB) carrying the2.4 kb BamHI-XhoI insert from pMammA, deposited with the American TypeCulture Collection in 10801 University Boulevard, Manassa, Va.20110-2209 on Feb. 22, 1996, and was given Accession No. 97451. (SeeExample 5.)

An expression vector containing the specific target DNA sequence is usedto generate all or a portion of Mammastatin protein, by in vitrotranscription and translation by insertion into cellular hosts forprotein production. Proteins produced from the expression vector systeminhibit the growth of mammary cells, normal and cancerous (See Example7.) Eucaryotic cells, e.g., Cos7 host cells, transfected with the vectorexpress and secrete Mammastatin into the conditioned medium. Conditionedmedium inhibited the growth of normal and cancerous mammary cells. (SeeExample 8.)

Amino Acid Sequence

The Mammastatin protein is a polypeptide having the sequence deducedfrom the nucleic acid sequence (SEQ ID NO.: 1) and shown in Table 1.Protein synthesized from the cloned Mammastatin nucleic acid sequence(SEQ ID NO.:1) inhibits breast cancer cell (MCF-7) growth.

Recombinant Mammastatin protein can be efficiently produced in purifiedform and in large quantities. Purified recombinant Mammastatin is usefulas a reliable standard for diagnostic assays of the inhibitor in patientsamples. Recombinant Mammastatin protein is also useful as a purifiedtherapeutic agent to inhibit or prevent the growth of breast cancercells.

Therapeutic Use

Mammastatin protein for therapeutic use is produced from NHMC culturesunder serum free conditions or by recombinant means. Mammastatinphosphoprotein is used therapeutically to inhibit mammary cell growth,e.g., in the treatment of breast cancer. Preferably, Mammastatin isproduced in higher eucaryotic cells to achieve phosphorylation of theprotein. Recombinant Mammastatin protein is produced in host cells or bysynthetic means.

Functional Mammastatin is administered to patients by known methods, forthe administration of phosphoprotein, preferably by injection, toincrease inhibitor levels in the bloodstream and increase theinhibitor's interactions with mammary cells.

The protein may be delivered to the patient by methods known in thefield for delivery of phosphorylated protein therapeutic agents. Ingeneral, the inhibitor is mixed with a delivery vehicle and administeredby injection.

The dosage of inhibitor to be administered may be determined by oneskilled in the art, and will vary with the type of treatment modalityand extent of disease. Since Mammastatin inhibits approximately 50% ofmammary cancer cell growth at a concentration of 10 ng/ml and stopsgrowth at about 20-25 ng/ml in vitro, a useful therapeutic dosage rangeis about 2.5 ug to about 250 ug administered daily dose. Preferred isapproximately 125 ug daily administered dose. The aim of theadministration is to result in a final body dose that is in thephysiological or slightly higher range (50-75 ng/ml). Higher doses ofinhibitor (>50 ng/ml) appear to induce apoptosis, as seen in histologyof treated cells. For clinical use, the preferred dosage range is about500 ng/ml for initial treatment of metastatic disease, followed by amaintenance dosage of about 50 ng/ml. Initial clinical studies, reportedin the examples below, indicate an administered daily dose of about 50ng/ml to about 750 ng/ml is sufficient to induce remission in Stage IVbreast cancer patients.

Since active Mammastatin is a phosphorylated protein, it is anticipatedthat multiple doses of the inhibitor will be required to maintain growthinhibiting levels of Mammastatin in the patient's blood. Also, sinceMammastatin generally acts as a cytostatic agent rather than a cytocidalagent, it is expected that a maximum effect of the inhibitor willrequire regular maintenance of inhibitor levels in breast cancerpatients.

In its preferred use, Mammastatin is administered in high dosages (>50ng/ml, preferably about 50-500 ng/ml) to induce tumor regression. Lower,maintenance doses (<50 ng/ml, preferably 20-50 ng/ml) are used toprevent cancer cell growth.

Clinical experience with administered Mammastatin in Stage IV breastcancer patients indicates a useful dose is that which maintainsphysiological levels of Mammastatin in the blood. Administration ispreferably daily, but, may be, for example, by continuous infusion, byslow release depot, or by injection once every 2-3 days. Anecdotalevidence suggests continuous administration may induce feedbackinhibition, thus, a preferred administration scheme is to administerdaily dose of Mammastatin for approximately 25-28 days, followed by 2-5days without administration.

Diagnostic Use

Assays of the present invention for detecting the presence of thefunctional inhibitor in human tissue and serum are useful in screeningpatients for breast cancer, for screening the population for those athigh risk of developing breast cancer, for detecting early onset ofbreast cancer, and for monitoring patient levels of inhibitor duringtreatment. For example, analysis of a patient's blood Mammastatin mayindicate a reduced amount of high molecular weight, phosphorylatedMammastatin, as compared with a normal control or with the patient'sprior Mammastatin profile. Such a change is correlated with increasedrisk of breast cancer, with early onset of breast cancer, and withadvancing metastatic breast cancer. Diagnostic assay for phosphorylated,active, 49/53 kD Mammastatin preferably is by Western blot immunoassay,e.g. ELISA, or using specific anti-Mammastatin antibodies. Screening,for example, in serum, is preferably by immunoassay, e.g., dot blotassay.

For best results, the patient samples should be assayed within a shorttime of sampling (within one week), stored at 4° C. (less than oneyear), or frozen for long term storage. Most preferably, samples arefrozen until time of assay.

Assay Kit

In a specific embodiment of the invention, an assay kit for thedetection of Mammastatin in a patient's fluid and/or breast tissue isprovided. The preferred screening assay is an immunoassay such as a dotblot assay to detect or quantitate Mammastatin in blood serum. Such ascreening kit includes anti-Mammastatin antibodies and optionally acontrol antibody and/or Mammastatin controls or standards. A secondscreening assay analyzes Mammastatin in breast tissue. Preferably, theassay kit contains necessary reagents and tools for reacting the tissuewith an antibody to specifically determine that the tissue is breastepithelium, e.g., an anti-cytokeratin antibody, and a specificanti-Mammastatin antibody. The commercially available antibody mixture,pan-keratin (Sigma) is a preferred anti-cytokeratin antibody.

A negative assay for Mammastatin could be caused by either the presenceof a breast cancer tumor, or by non-epithelial breast tissue. Use of theanti-cytokeratin antibody guards against false positive assays.Epithelial cells of the breast that do not stain with theanti-Mammastatin antibody or which only express the 44 kD Mammastatinare transformed cells. Thus, by first identifying the tissue as breastepithelium, e.g., isolated from breast tissue and positive with theanti-cytokeratin antibody, and then identifying a second positivereaction with anti-Mammastatin antibody, false positives are avoided.

Because about 30% of the breast cancer cells studied to date expressnon-phosphorylated inactive, 44 kD Mammastatin, the preferred method ofanalysis is to differentiate between the 53/49 kD and 44 kD forms, e.g.by Western blot analysis.

The invention is further defined by reference to the following examples:

EXAMPLE 1 Human Mammary Cell cDNA Library

A cDNA library was prepared from human mammary cells obtained fromreduction mammoplasties (UM Hospital). Total RNA was isolated from themammary cells by cesium chloride gradient. From the total RNApreparation, mRNA was isolated. The methods used were those described inGarner I., “Isolation of total and poly A+ RNA from animal cells”,Methods Mol. Biol. (1994) 28:41-7.

Reverse transcriptase in the presence of the isolated mRNA produced cDNAthat was then ligated to EcoRI linkers. The cDNA was inserted into EcoRIcut T4 DNA ligase-treated Lambda Zap, and amplified in XL1-blue E coli,following the method described in Short J M., et al. (1988) NucleicAcids Research 16: 7583.

EXAMPLE 2 Preparation of Mammastatin Oligonucleotides

The normal human mammary cell cDNA library prepared in Example 1 wasscreened for the presence of nucleic acids encoding Mammastatin usingdegenerate oligonucleotides. The degenerate oligonucleotides werederived as follows:

Normal human mammary cells were obtained from the Plastic SurgeryDepartment of the University of Michigan Hospital or from theCooperative Human Tissue Network. The tissue was reduced by collagenasetreatment generally following the procedure described in Soule, et al.,In Vitro, 22:6 (1986).

Mammary cells were grown to confluence in 175 cm² flasks in DMEM/F12 lowcalcium media formulated with 40 μM CaCl₂ and supplemented with 5%CHELEX treated equine serum (Sigma), 0.1 μg/ml cholera toxin (Sigma),0.5 μg/ml hydrocortisone (Sigma), 10 ng/ml epidermal growth factor (EGF,Collaborative Research, Bedford Mass.), 10 μg/ml insulin, and 1 μg/mlpenicillin/streptomycin following the method described in Soule, et al.,In vitro 22:6(1986). Equine serum was treated with CHELEX resin forthree hours at room temperature to remove serum calcium.

Cell lysates were prepared by rinsing cells with TBS and scraping fromthe flask with a Teflon scraper. Cells were collected by centrifugationand lysed with 8M Urea, 50 mM TRIS pH 7.5, 0.5% Beta-mercaptoethanol,0.5% TRITON X-100 (lysis buffer) and three minutes of sonication on ice.

The cell lysates were fractionated on DEAE-Sephacel anion exchange resin(Sigma) equilibrated with lysis buffer. Lysates were loaded onto theresin filled columns (50 ml disposable, Bio Rad) and washed with tencolumn volumes of the lysis buffer. Material flowed through the columnswith only gravity feed. Fractions were eluted with a salt gradientproduced by continuous gravity feed of elution buffer containing 5M NaClinto a closed mixing chamber initially containing elution buffer (250 mlof 8M urea and 50 mM TRIS pH 7.5) in the absence of salt.

Elution fractions (2 ml) were collected with a Gibson fractioncollector, and were analyzed for the presence of mammary cell growthinhibitor by dot blot with the anti-Mammastatin antibody, 7G6, describedabove.

Positive fractions were pooled and dialyzed into lysis buffer with 50 mMNaCl, and were again separated on an identical ion exchange column andeluted with a continuous decreasing pH gradient (pH 8 to pH 3) inelution buffer with 50 mM NaCl. (To produce the pH gradient, pH 3buffered urea was continuously mixed with the initial pH 8 buffer.)Fractions (2 ml) were collected and analyzed with the 7G6 antibody asdescribed above.

Positive fractions were again pooled and concentrated to 1/10 theoriginal volume by filtered centrifugation (Amicon Centriprep, 10 kDcutoff). The concentrated pool was size fractionated by preparative SDSpolyacrylamide gel electrophoresis (PAGE) along with prestainedmolecular weight standards (Sigma).

Protein contained in the molecular weight range between 40 and 60 kD wasexcised from the gel in 0.5 cm strips or fractions. Electroelution ofthe protein from each gel strip was carried out by placing the gel stripin 1 ml of running buffer (192 mM glycine, 25 mM TRIS pH 8.3, 0.1% SDS)in dialysis tubing. The tubing was placed in a submarine electrophoresisapparatus and electroeluted overnight at 25 volts. Current was reversedfor 2 minutes and running buffer, now containing the electroelutedprotein, was removed. Purity of the eluted protein was checked byanalytic SDS PAGE with silver-staining, and also by immunoblot with the7G6 antibody, following the procedure described in Towbin et al., J.Clin. Chem. Clin. Biochem. 27:495-501 (1989). Fractions that were atleast 70% pure as determined by silver-stained PAGE were pooled,concentrated, and lyophilized to powder form.

The pooled protein was cleaved with cyanogen bromide by resuspendinglyophilized powder in 500 μl of 70% formic acid and incubating overnightat room temperature (about 20 hours) with 20 mg/ml of cyanogen bromide(Sigma). The methods used are described in Freemont, et al., Arch.Biochem. Biophys. 228:342-352 (1986). Cyanogen bromide-cleaved proteinsamples were dialyzed into double distilled, deionized water and againconcentrated and lyophilized to powder.

Cyanogen bromide cleavage generated multiple peptides from the originalprotein sample, which were separated by preparative 15% SDS PAGE andtransferred onto PVDF membrane by electroelution.

In addition to the protein obtained from mammary cell lysates, proteinwas also isolated from normal human mammary cell conditioned medium.Normal cells were incubated with 8 ml DMEM lacking phosphates andsupplemented with 200 μCi/ml ³²P-ortho-phosphate and 1% dialyzed fetalbovine sera. Cells were allowed to grow for 24 hours in the presence ofthe ³²P before conditioned media was collected.

The collected conditioned media was concentrated 5× by Amicon filtrationwith 10 kD exclusion limit. Concentrated media was rinsed once with PBSon filtration membranes to remove excess unincorporated phosphate andwas further fractionated by S-200 SEPHACRYL (Pharmacia, Upsala, Sweden)molecular sieve chromatography (100 cm×0.75 cm column) eluted with PBS.Both the filter and the column permit removal of unincorporated ³²P fromthe sample. One ml fractions were collected from the column, and labeledfractions identified by scintillation counting. Radioactive fractionswere pooled and analyzed by SDS PAGE with silver staining andautoradiography. The pooled protein was concentrated, lyophilized topowder, and combined with the larger mass of unlabeled protein purifiedas described above, before cyanogen bromide cleavage. The addition oflabeled protein provided a convenient means of tracing cyanogen bromidecleavage fragments containing phosphorylated Mammastatin peptides.Cleaved peptides were separated on preparative PAGE as described above.

After radioactive proteins were cyanogen bromide cleaved, separated,transferred to PVDF membrane, and exposed to X-ray film, two labeledbands of approximately 20 and 22 kD were seen. These two peptides wereexcised from membranes and sequenced by Edman degradation methods at theUniversity of Michigan Biomedical Research Core Facility using methodsdescribed in Ullah Alt et. all., Biochem. Biophys. Res. Comm.203:182-189 (1994). The amino acid sequences of each of the two peptideswere compared with known database sequences using the NIH “BLAST”server. The two peptides appeared to be unique.

A particularly unique portion of each sequence was used to producedegenerate oligonucleotides, using the standard third positiondegeneracy according to the method described in Jerala, Biotechniques13:564-567 (1992). From the 20 kD peptide, the sequence“gly-gln-leu-glu-tyr-gln-asp-leu-arg” (SEQ ID NO.:2) was used; from the22 kD peptide, the sequence“tyr-glu-arg-asp-leu-lys-gly-arg-asp-pro-val-ala-ala” (SEQ ID NO.:3) wasused to generate multiple species of oligonucleotides. The degenerateoligonucleotides were purified by high pressure liquid chromatography.

SEQ ID NO.: Peptide 2 gly gln leu glu tyr gln asp leu arg 3 tyr glu argasp leu lys gly arg asp pro val ala ala

The degenerate oligonucleotides were end-labeled with ³²P-gamma ATP andT4 DNA polynucleotide kinase (BRL, Bethesda, Md.) and resuspended in T4DNA kinase buffer (60 mM TRIS pH 7.8, 10 mM MgCl₂, 15 mMbeta-mercaptoethanol) at 1.5 mg/ml. Oligonucleotides (250 μM) were thenincubated with 0.33 μM ATP, 5 units kinase in 25 μl kinase buffer, fortwo hours at 37° C. Incorporation of ³²P-phosphate was determined by TCAprecipitation (15% TCA, 4° C., 15 minutes). Typical incorporation was10⁹ cpm/μg DNA.

EXAMPLE 3 Screening Mammary Cell CDNA Library with DegenerateOligonucleotides

Bacteria infected with phage prepared for Example 1, containing a normalmammary cell cDNA insert, were plated on 15 cm NZCYM (10 g, NZ amine(Bohringer Manheim), 5 g NaCl, 5 g yeast extract, 2 g MgSO₄, 1 gcasamino acids) plates in top agar ( 1/10 dilution of infected bacterialcultures to 6 ml of 7% NZYM top agar) and allowed to incubate eighthours at 37° C. Plates containing plaques were overlaid withnitrocellulose for 15 minutes before denaturation of phage. Phage wasdenatured by blotting filters (DNA side up) on Whatman paper saturatedwith 0.5 M NaOH, 1.5 M NaCl for 5 minutes. Filters were rinsed with H₂Obefore incubating for 5 minutes in 1 M TRIS pH 7.0, 1.5 M NaCl followedby 20× SSC and 2× SSC, each for 5 minutes. Filters were dried and bakedfor 1 hour at 80° C. or placed under ultraviolet light to immobilizeDNA. Baked filters were washed for 30 minutes in 2× SSC with 1% SDS andthen prehybridized with 50% deionized formamide, 5× Denhart's solution,1% SDS, 5× SSC and 100 μg/ml sheared salmon sperm DNA overnight at 37°C.

Filters were hybridized with the labeled degenerate oligonueleotideprepared as described for Example 2 in prehybridization buffer to which10⁷ cpm/ml of heat-denatured (95° C., 5 minutes) labeled degenerateoligonucleotide had been added. Hybridizations were performed at 37° C.for 24 hours. Filters were washed with 2× SSC for thirty minutes at 37°C. followed by 3 washes in 2× SSC plus 1% SDS at 50° C. for thirtyminutes. Filters were rinsed with 2× SSC briefly, dried and exposed toKodak AR-5 film for 24-48 hours to identify positive plaques.

Positive plaques were isolated from agar plugs excised using a reversed200 μl sterile pipette tip, and resuspended in SM buffer overnight at 4°C. Secondary and tertiary plates (10 cm) were made using XL1-B infectedwith 1/10,000 dilution of phage containing SM buffer, to bacteria, inNZCYM (with 1 mM MgSO4). Plaques were produced by incubating infectedbacteria for 8 hours as described above, and were then transferred tonitrocellulose before screening with labeled degenerateoligonucleotides. Screening was performed essentially as described inKroczek R A., J Chromatogr 618:133-45(1993), using 10⁷ cpm/ml of labeledDNA for hybridizations and a final wash stringency of 2× SSC at 50° C.for thirty minutes.

The clone selected for further analysis was one recognized by both ofthe degenerate oligonucleotides. This clone was given the name “pMammA”.

EXAMPLE 4 Sequencing of Mammastatin cDNA

The positive clone obtained in Example 3, pMammA, was sequenced by anautomated sequencer at the Biomedical Research Core Facility at theUniversity of Michigan and also by dideoxy DNA sequencing using 15% DNAsequencing gels and radiolabeling the DNA fragments with ³⁵Snucleotides. The methods used are described in Lasken R S., et al. ProcNatl Acad Sci U S A 82:1301-5 (1985). The nucleic acid sequence obtainedis shown below in Table 1 (SEQ ID NO.: 1).

The recognized error rate of automatic sequences is about 5%. Therefore,the clone deposited is resequenced for confirmation of the nucleotidesequence, particularly mindful of areas suspected of potential errors,as noted.

EXAMPLE 5 Subcloning the Mammastatin cDNA into an Expression Vector

The Mammastatin cDNA insert, pMammA, was subcloned into the expressionvector, pcDNA 3 (Invitrogen). The Mammastatin cDNA was isolated bydigesting the pMammA plasmid obtained as described for Example 4 withBamHI and XhoI restriction endonucleases. The restriction enzymes cutthe plasmid at the ends of the Mammastatin clone insert, creating alinear plasmid fragment and a linear insert fragment. The digestedsample was placed in the wells of a 1.2% agarose gel submerged in anelectrophoresis apparatus, a 50V current was applied for two hours.Electrophoresis separates DNA fragments on the basis of size with thelarger plasmid DNA fragment having the slower migration rate on the gel.The portion of the agarose gel containing the 2.4 kb was visualized byethidium bromide staining and observing the gel over an ultra-violetlight box. The 2.4 kb Mammastatin fragment was cut from the gel andplaced into dialysis tubing and the DNA was electrocluted intotris-borate buffer, TBE: (0.089M Tris-borate, 0.089M boric acid, 0.002MEDTA) that was collected and precipitated with ethanol.

The pcDNA3 plasmid DNA was modified to accept the Mammastatin cDNAfragment during ligation. pcDNA3 plasmid was digested with BamHI andXhoI restriction endonucleases and after digestion was complete, the DNAwas incubated for one hour in the presence of calf intestinalphosphatase to remove 5′ phosphates. The pcDNA3 sample was then phenolextracted and ethanol precipitated.

The pcDNA3 and the Mammastatin 2.4 kB cDNA fragment were ligatedtogether. The 2.4 kb Mammastatin fragment and the linear pcDNA3 plasmidwere mixed in a 3:1 ratio in the presence of T4 DNA ligase. The ligationreaction was allowed to incubate for one hour and then stored at 4° C.overnight. After the ligation reaction was completed the DNA was used totransform E. coli competent cells. Subcloning was verified by purifyingplasmid DNA from ampicillin selected colonies. The plasmids weredigested with the restriction endonucleases BamHI and XhoI. The digestedDNA samples were placed in an agarose gel and separated byelectrophoresis. A plasmid containing the correct size Mammastatin DNAfragment was designated pMammB, and was deposited with the American TypeCulture Collection (ATCC) on Feb. 22, 1996, and given accession number:ATCC 97451.

EXAMPLE 6 Transfection and Protein Expression from the Mammastatin cDNASequence

Cos-7 cells do not express immunoreactive proteins that co-migrate withthe Mammastatin proteins. pMammB and PcDNA3 were used to transfect Cos-7monkey fibroblast cells using LIPOFECTIN® (BRL, Life Technologies,Bethesda, Md.) using the manufacturers suggested protocol. Thetransfected cells were grown for two days prior to harvest. Transfectedcells were removed from plates by trypsinizaton of cells using standardprotocols. (2.5 mls of Trypsin (0.25% SIGMA) was incubated in flasks ofcells at 37° C. for 5 minutes. A 7.5 ml aliquot of RPMI media with 10%FBS (fetal bovine serum) was added and cells were collected bycentrifugation.) Cells were counted by hemocytometer and lysed in SDSPAGE sample loading buffer at 10⁷ cells/ml. Cell lysates were separatedon 8-15% SDS-PAGE gradient gels (Biorad) and transferred to a nylonmembrane using methods described in Towbin H., et al., J. Clin Chem ClinBiochem (1989 Aug.) 27(8):495-501. The membrane was probed withanti-Mammastatin monoclonal antibody 7G6. Bound antibody was detectedwith peroxidase conjugated GAM-IgM and developed by ECL (Amersham).

As shown in FIG. 1, Cos-7 cells transfected with pMammB (lanes C,D)expressed immunoreactive proteins that co-migrated with Mammastatinprotein (lane A). Cos-7 cells transfected with the empty vector PCDNA3alone did not express immunoreactive proteins when immunoblotexperiments were performed (lane B).

DNA/AA SEQUENCE Lane A NHMC (25 μg) - control Lane B Cos pcDNA3 celllysate (25 μg) - control Lane C Cos-pMammB cell lysate (10 μg) Lane DCos-pMammB cell lysate (20 μg)

The immunoblot experiments illustrate the pMammB clone contains a cDNAinsert capable of synthesizing a protein with the size and immunologiccharacteristics of Mammastatin. In addition, immunoreactive proteins of44, 49 and 53 kD were expressed in Cos-7 cells transfected with pMammB.These proteins migrated at the same molecular weight as the Mammastatinproteins previously identified in normal human mammary cells. This groupof immunoreactive proteins was not identified in Cos-7 cells transfectedwith the empty vector, pcDNA3.

In the particular assay shown in FIG. 1, the NHMC control shows anunusually high amount of 44 kD Mammastatin. This is an artifact producedby long term (>1 yr) storage of the NHMC standard at 4° C., causingdegradation of the higher molecular weight forms, over time. Whenfresher NHMC samples (<1 yr old) or frozen samples are used, the 44 kDprotein is always less abundant than the higher molecular weight forms.

EXAMPLE 7 GST Fusion

The Mammastatin clone can be similarly subcloned into a baculovirusexpression system. The pMammA insert has been subcloned into a pAcG3Xvector obtained commercially from Pharmingen (San Diego, Calif.). Thisvector allows production of Mammastatin as a fusion protein withglutathione S-transferase (GST), having a portion of the GST geneupstream of the coding site.

The pMammA insert was subcloned by preparing sets of PCR primers thatcontained BamHI (5′) and SmaI (3′) restriction enzyme recognition sites,a small, non-specific region, and a portion of the Mammastatin sequence.Three sets of primers, each shifted in reading frame, were prepared. Theprimers hybridized to the pMammA clones and in a typical PCR reactionwith pMammA template DNA, amplified a pMammA PCR product capable ofinsertion into the reading frame of the GST gene in pAcG3X. The vectorwas then used to transfect High 5 (Invitrogen) host insect cells, andexpress a GST-Mammastatin fusion protein that was easily purified fromhost insect cells using glutathione resin (glutathione agarose, Qiagen,Chatsworth, Calif.).

To prepare DNA for insertion into the BamHI, SmaI restriction site ofpAcG3X (PharMingen, San Diego, Calif.), primer sets were prepared inthree reading frames to include, for the 5′ primer, the BamH1recognition site (GGATCC), a portion of the pMammA sequence, and some 5′sequence from the pBluescript vector. The 3′ primers were identical, andincluded the SmaI recognition sequence (GGG CCC), a portion of thepMammA sequence, and some pBluescript sequence.

The primer sets used are shown in the following table:

SEQ ID NO.: 5′ Primers (in three reading frames)* 4 5′-TGG GAT CCC TTCGCC ACG AGC ACG GTG-3′ 5 5′-TGG GAT CCT TCG CCA CGA GCA CGG-3′ 65′-TGG GAT CCC CTT CGC CAC GAG CAC-3′ 3′ Primer 7 5′-TTT TTT TTT TTTGGG CCC TTA AGT-3′** *BamHI site underlined **SmaI site underlined

Only one primer set (SEQ ID NOS.:5 and 7) produced clones capable ofcoding for active inhibitory Mammastatin. The active clones, when usedto transform High 5 cells, produced Mammastatin that was immunologicallyreactive in the transformed cells (see FIG. 2).

Other known eukaryotic expression systems may similarly be used toproduce Mammastatin protein.

EXAMPLE 8 Inhibition Assay with Proteins Produced by In VitroTranscription and Translation

In vitro transcription of pMammB, Mammastatin cDNA was performed using aStratagene Express RNA transcription kit to produce Mammastatin RNA. TheRNA produced was translated into protein using the Stratagene In VitroExpress translation kit (see FIG. 3A). Mammastatin protein produced fromtranslation of the Mammastatin RNA was shown to inhibit mammary cellgrowth in culture.

Cultures of MCF-7 cells were treated with protein products produced inthe translation assays described above. Protein products (5% by volume,culture medium) were added to cells in 12-well plates containing 1 mlmedium per well. Parallel cultures were treated with both thetranslation product and the anti-Mammastatin antibody 3C6, at 30 μg/mlfinal concentration.

As a negative control, cultures were treated with protein productstranslated with the Stragene In Vitro Express Translation kit incubatedin the absence of Mammastatin cDNA (i.e. employ vector). These lysatesdo not have the proper machinery to produce the Mammastatin protein.

All cultures were allowed to grow for six days after being treated withthe protein products and the cell number of each sample was calculatedusing a Coulter counter. There were triplicate samples of each culturecondition so that the cell number of each sample was averaged andpercent inhibition was determined by comparison to the reticulocytelysate treated control cells.

As shown in FIG. 3B, the protein translation product of pMammB inhibitedMCF-7 cell growth. This inhibition was greatly reduced or blocked in thepresence of anti-Mammastatin antibody, 3C6.

EXAMPLE 9 Inhibition Of Mammary Cells with Proteins Present WithinConditioned Media Obtained From Growing Cos-7 Cells Transfected WithpMammB

Mammary cell growth inhibition experiments were performed usingconditioned media obtained from Cos-7 cells transfected with pMammB asdescribed for Example 6. Mammastatin is a secreted protein and is foundin conditioned media of cells expressing the protein. The growthinhibition caused by conditioned media was blocked by the addition ofanti-Mammastatin antibody.

MCF-7 cells were plated at 10⁴ cells/ml in MEM supplemented with 10%non-essential amino acids and FBS (SIGMA). Cells were allowed to attachovernight and were then supplemented with 10% by volume of conditionedmedia (3 day culture) from either: (1) Cos-7 cells transfected with theempty vector pcDNA (Negative control), (2) Cos-7 cells transfected withpMammB (pMammB-Cos), (3) NHMC-conditioned media, or (4) non-conditionedmedia. Parallel MCF-7 cultures were supplemented with 30 ug/ml of 3C6blocking antibody. Treated MCF-7 cells were allowed to grow for six daysand were then counted by hemocytometer.

Inhibition of cell growth was determined by comparing the growth ofMCF-7 cells incubated in conditioned media with the growth of MCF-7cells incubated in control, non-conditioned media. Data are shown inFIG. 4, and demonstrate that conditioned media from pMammB-transformedcells inhibited mammary cancer cell growth as efficiently as did normalhuman mammary cell conditioned media. This inhibition was blocked in thepresence of anti-Mammastatin antibody.

EXAMPLE 10 Three Immunologically Reactive Anti-Mammastatin Proteins

Whole normal human mammary cells (NHMC) and mammary carcinoma cells intissue culture cells were lysed, and cell lysate proteins were separatedby SDS/PAGE as described above and in Ervin, Paul, 1995, Doctoraldissertation, University of Michigan, Chapter 2. Lysed cell samples wereseparated on 10% SDS-PAGE in a Mini-Protean II apparatus (25 μg/sample).Proteins were transferred to nitrocellulose and probed with theanti-Mammastatin monoclonal antibody 7G6 or IgM control antibody,alkaline phosphatase conjugated second antibody, goat anti-mouse IgM wasutilized with an NBT/BCIP substrate system to detect positive antibodyreactions colorometrically. The data are shown in FIG. 5.

CARCINOMA CELLS LANE 1 ZR-75-1 LANE 2 MDA MB 435 LANE 3 4MCF-7 LANE 4T47D LANE 5 NHMC-14 positive control LANE 6 NHMC-14 positive controlwith the 38C13 antibody NORMAL CELLS LANE 7 NHMC-17 LANE 8 NHMC-16 LANE9 NHMC-15 LANE 10 NHMC-14 LANE 11 NHMC-6 LANE 12 NHMC-14 positivecontrol

As shown in FIG. 5, normal human mammary cells expressed a doublet ofproteins migrating at 49 and 53 kD that were strongly recognized by theanti-Mammastatin monoclonal antibody and a third weakly immuno-reactive44 kD protein. The four tumor cell lines tested expressed either a 44 kDimmuno-reactive protein alone (lanes 1,4) or no immunoreactive proteinat all (lanes 2, 3).

The above data is representative of experiments performed on normalcells from 42 different reduction mammoplasty patients over a period ofseveral years. Expression of the 44 kD protein in normal cells andcancer cell lines varied in intensity with each preparation.

EXAMPLE 11 Mammastatin is a Phosphoprotein

Cellular phosphorylated proteins of mammary cells were labeled with ³²Pby supplementing normal mammary cell cultures with ³²P-orthophosphate(200 μCi/ml) for 24 hours. Conditioned media was concentrated 5× byAmicom Centrifugation with a 30 kD molecular weight restriction.Concentrated media was rinsed once with PBS on filtration membranes toremove excess unincorporated phosphate and fractionated by S-200SEPHACRYL (Pharmacia, Upsala, Sweden) molecular sieve chromatography(100 cm×0.75 cm column) with PBS elution buffer. Immunoblots wereprepared as described above and probed with the 7G6 antibody.

A radiolabeled 53 kD Mammastatin protein was identified in conditionedmedia by immunoprecipitation. This analysis indicated Mammastatin is asecreted phosphoprotein. Since secreted phosphoproteins are uncommon,Brefeldin A treatment of cells was utilized to determine whetherMammastatin was present in conditioned media due to secretion or to cellbreakage or leaking. Brefeldin A is a fungal compound that blocks thesecretion of proteins from eukaryotic cells. Brefeldin A inhibits normalendoplasmic reticulum and golgi function and blocks vesicle formation(Ervin, Paul, 1995, Dissertation, Page 25). Since most secreted proteinsare liberated from the cell by a process of exocytosis from membranebound vesicles, blocking vesicle formation blocks secretion of manyproteins. When NHMC are grown in the presence of Brefeldin A,phosphorylated Mammastatin is not identified in conditioned media.

To determine the amino acid residues that are phosphorylated inMammastatin protein, radiolabeled 53 kD protein was subjected tophospho-amino acid analysis. NHMC cells were incubated with³²P-orthophosphate for 24 hours. Cell lysates were thenimmunoprecipitated with the anti-Mammastatin antibody 7G6 and purifiedas follows. The 53 kD protein was digested with trypsin and hydrolyzedwith acid. Two dimensional thin layer chromatography was used to analyzethe phosphorylated amino acids of Mammastatin. ³²P-amino acids weremixed with phospho-ser/thr/tyr controls and loaded at the origin (0) ofa 2D TLC plate (20 cm). The samples were separated into two dimensions:1st dimension—pH 1.9 Buffer (50 ml formic acid, 156 ml glacial aceticacid/2000 ml (1794H₂O), 20 minutes @ 1.5 K volts; rotate clockwise; 2nddimension—pH 3.5 Buffer (10 ml pyridine, 100 ml's glacial acetic acid:1890 ml H₂O) for 16 minutes @ 1.3 K volts.

The TLC plates were stained with ninhydrin and exposed to film.Phospho-amino acid analysis demonstrated the 53 kD Mammastatin proteincontained three types of phosphorylated amino acid residues by comparingautoradiographs to ninhydrin stained phospho-amino acid standards.

Threonine (Th) was the most abundant phosphorylated amino acid followedby serine (S) and Tyrosine (Ty), the least abundant phosphorylatedspecies. However, the relative abundance of phosphoamino acid residuesmay not be representative of that in the native protein, since acidhydrolysis can free phosphate from phosphotyrosyl residues.

EXAMPLE 12 One Mammastatin Protein with Varied Phosphorylation

Cellular phosphorylation of proteins can be modulated by phosphatasesand kinases. Mammastatin is differentially phosphorylated in normal andtumor cell lysates due to differential activities of Mammastatinphosphatases. The effect of phosphatase on Mammastatin in NHMC lysateswas examined.

NHMC were grown to confluence in low calcium media and collected byscraping into TBS. Cells were washed with TBS and resuspended at 2 mg/mlin acetate buffer pH 6.6 with 0.5% Triton X-100. 5 μg/ml of eitherYersinia phosphatase (YOP)(Stuckey, et al., Nature 370:571-5 (1994)) orYersinia phosphatase mutant (MYOP) containing an active site mutationwas used to digest cell lysates for six hours at 37° C. (YOP and MYOPwere gifts from Dr. S. Jack Dixon, University of Michigan, BiochemistryDepartment). As shown in FIG. 6, digestion of normal human mammary celllysates with Yersinia phosphatase (YOP) resulted in a reduced amount of53 kD Mammastatin protein identified by anti-Mammastatin immunoblot(lane A). In contrast, digestion with the Yersinia phosphatase mutant(MYOP, lane B), did not alter identification of the 53 kD Mammastatinprotein. These results indicate identification of the 53 kD Mammastatinprotein by immunoblot is a convenient measure of the state ofphosphorylation of the Mammastatin protein.

Conditioned medium incubated in the presence of Yersinia phosphatase(YOP), as described above, was used to treat MCF-7 cells. As previouslyobserved, NHMC conditioned medium inhibits the growth of MCF-7 cells,and this inhibition is blocked by anti-Mammastatin antibodies. As shownin FIG. 7, treatment of NHMC conditioned medium with YOP abrogates thisinhibitory activity. As a control, treatment of NHMC conditioned mediawith a YOP mutant lacking phosphatase activity (M.YOP) was tested. Thismutant had no effect on the inhibitory activity of NHMC conditionedmedia. Immunoprecipitation of the conditioned media with theanti-Mammastatin antibody 7G6 removed the inhibitory activity.

TCA precipitation indicated that incubation of conditioned media withYOP removed about 50% of incorporated phosphate. As shown above, YOPalso removed the 53 kD species from NHMC lysates (FIG. 6).

EXAMPLE 13 Phosphorylated Mammastatin Produced by Normal But NotCancerous Mammary Cells

Normal and transformed mammary cells were labeled with ³²Porthophosphate. Carcinoma cell lines were grown in the media assuggested by the ATCC, with the exception of MCF-7 cells which weregrown in MEM (Celox) supplemented with 10% FBS, non-essential aminoacids, and insulin (10 mg/l). ³²P-orthophosphate labeling of cellularproteins was performed in phosphate-free DMEM (ICN) containing 2%dialyzed FBS. Cells were incubated 24 hours at 37° C. with 200 μCi/ml of³²P-phosphate. After 48 hours, conditioned media was collected from cellcultures and concentrated 5×. Conditioned media was washed with TBS andconcentrated on Amicon filters with a 10 kD mw cut-off. The cell layerwas scraped (using a Teflon cell scraper) into lysis buffer, 1.5ml/flask (0.5% Triton X-100, 2.01% SDS at deoxycholate) from celllysates and conditioned media.

Mammastatin proteins were immunoprecipitated by adding 5 μg 7G6anti-Mammastatin antibody per 500 μl of 5× concentrated media or celllysate and incubating at room temperature for 1.5 hours. Goat anti-mouseIgM second antibody (5 μg/0.5 ml) was added and the mixture incubated anadditional hour. Protein G PLUS/A agarose® slurry (Oncogene Science) wasadded and the mixture incubated 1.5 hours at room temperature toimmobilize antibody complexes.

The complexes were washed 6× with lysis buffer, each wash followed bycentrifugation at 3000× g. SDS-PAGE loading buffer (50 μl) was addedbefore the sample was heated to 100° C. for 3 minutes. Supernatants wereresolved by SDS-PAGE, transferred to nitrocellulose, and exposed toKodak X-AR film.

Phosphate labeling of NHMC proteins and subsequent immunoprecipitationidentified 49 and 53 kD phosphoproteins in NHMC. The 49 and 53 kDphosphoproteins were not recognized in carcinoma cell lines. Carcinomacell lines MCF-7, T47D, ZR-75-1 and MDA-MB-435 expressed a 44 kDimmunoreactive protein, but this protein did not label with³²P-orthophosphate.

This study indicates more incorporated phosphate with increasingmolecular weight of Mammastatin. Lack of phosphorylation of Mammastatinin transformed cell lines correlates with lack of higher molecularweight forms of the protein and lack of Mammastatin inhibitory activity.

EXAMPLE 14 Mammastatin Kinase & Phosphatase

Flasks of normal or carcinoma cells were grown to 75% confluence. Cellcultures were washed three times with TBS and then scraped into TBS witha Teflon scraper. Cell suspensions were pelleted at 1000 g bycentrifugation and then resuspended in a small volume of TBS. An aliquotof each type of cell was removed for protein quantitation. Proteinconcentrations were then equalized at 2 mg/ml in lysis buffer (TBS with0.5% Triton X-100 and 5 μg/ml each of aprotinin, leupeptin, and PMSF).Equal masses of normal and tumor cell proteins were mixed and incubatedat 37° C. for three hours. Parallel mixtures of normal and carcinomacell lysates were performed in the presence of 10 nM orthovanadate(NaVO₄), a phosphatase inhibitor. The mixture was then separated bySDS/PAGE and analyzed by Western Blot using the 7G6 antibody. The dataare shown in FIG. 8.

LANE A ZR-75-1 Lysate (30 μg) LANE B NHMC Lysate (30 μg) LANE C NHMC (30μg) + ZR 75 (30 μg) + 10 nM NaVO₄ LANE D NHMC (30 μg) + ZR 75 (30 μg)

As shown in FIG. 8, cancer cells (ZR-75-1)(lane A) did not produce 53/49kD Mammastatin, as compared with NHMC (lane B). Mixing of normal andcancer cell proteins, in the presence of proteinases, reduces the amountof active, 53 kD inhibitor (lane D). However, in the presence of thetyrosine-phosphatase inhibitor NaVO₄, the 53 kD species is retained inthe mix (lane C). These results indicate that carcinoma cells expressphosphatase activity capable of eliminating phosphorylated forms ofMammastatin.

Expression of Mammastatin in normal and transformed cell lines can bemeasured quantitatively by Western blot analysis. Using anti-Mammastatinmonoclonal antibodies, it has been demonstrated that there is aconsistent difference in expression of this protein between mammarycarcinoma cells and cells derived from normal mammary epithelium.Mammastatin was recognized in normal human female mammary tissue as 44,49, and 53 kD species by Western blot analysis with anti-Mammastatinmonoclonal antibody 7G6. In mammary carcinoma cells, there wasinconsistent recognition of a 44 kD species, but never 49 or 53 kDimmunoreactive forms. When the 49 and 53 kD forms are identified innormal cells they are phosphorylated. The 44 kD species is notphosphorylated. It is therefore possible to use immunoblot analysis todetermine if Mammastatin is phosphorylated by observing the expressionof the 44 and 49, and 53 kD species of Mammastatin.

EXAMPLE 15 Identification of Mammastatin in Human Sera

An enzyme-linked immunosorbant assay (ELISA) was established to detectMammastatin, using the purified anti-Mammastatin monoclonal antibodies6B8 and 3C6.

The antibody 6B8 was used to coat Immulon 1 96-well microtiter plates(Immulon Corp.) at a concentration of 10 μg/ml or 100 μl/well, for three(3) hours at room temperature, or overnight at 4° C. Plates were blockedwith 2% BSA (Sigma) in TBS (150 mM NaCl, 100 mM Tris pH 7.4) for 30minutes and were then incubated with either purified Mammastatin orsample sera diluted 50% in 2% BSA solution for 1.5 hours at 37° C.Microtiter plates were washed for 5 minutes, three times with 300μl/well of TBS plus 0.1% Triton X-100 before addition of secondantibody.

Second antibody was biotinylated 3C6. Antibody was biotinylated byincubation with biotin, N-hydroxy succinimate ester (Sigma) in 0.1 MNaHCO₃ for two hours at room temperature and 16 hours at 4° C. Antibodywas dialyzed into 1M NaCl, 50 mM Tris pH 7.4, 0.02% Azide (NaN₃, Sigma)before use or storage.

Biotinylated anti-Mammastatin antibody was added at 1 μg/ml and 100μl/well, in a 2% BSA/TBS solution and incubated for 1.5 hours at 37° C.Microtiter plates were washed 5 times for 5 minutes with TBS plus 0.1%Triton X-100 as described above. Second antibody was identified withalkaline phosphatase conjugated streptavidin (Southern Biotechnology)and incubated for one hour at a dilution of 1/1000 in 2% BSA/TBS, 100μl/well for all samples.

ELISA assays were developed colorometrically with PNPP (para-nitrophenylphosphate Sigma), 1 mg/ml in alkaline phosphatase buffer (10 mMdiethanolamine pH 9.5 (Sigma), 0.50 mM MgCl₂ (Sigma)). Microtitre plateswere read on an ELISA reader at 405 nm at fifteen minute and thirtyminute intervals.

Using chromatographically purified Mammastatin isolated from celllysates or conditioned media, a standard curve was established for theELISA indicating sensitivity of the assay for Mammastatin in the lownanogram range. (See FIG. 9). Quantitation of Mammastatin levels innormal human volunteer sera was performed in serum samples collected attwo day intervals for one month, from a volunteer. Mammastatin levels innormal human female sera were detectable by this assay and variedbetween about 10 and 50 ng/ml (FIG. 10).

Mammastatin levels were also measured in sera collected from breastcancer patients. Patients diagnosed at the University of Michigan BreastCare Center with node negative breast cancer were tested for Mammastatinexpression in sera throughout the course of their treatment. The dataare shown in FIG. 11 and summarized below. Serum samples were collectedfrom breast cancer patients during the entire course of their treatmenton a hormonal cycling, combined modality protocol with Cytoxin,Adriamycin, Methotrexate, and 5 Fu. Serum was separated from wholeblood, after clotting, by centrifugation and stored at −20° C. untiluse. ELISA assay using 150 μl serum at 50% in 0.5% NFDM in duplicatewere performed, using the 6B8 and 3C6 anti-Mammastatin antibodies in anenzyme linked—“sandwich” assay. The standard curve was generated withchromatographically purified Mammastatin and was comparable to thatshown in FIG. 9.

Expression of Mammastatin varied among patients and fluctuated duringthe course of their treatment. It was consistently observed thatMammastatin levels became undetectable with progression to metastaticdisease.

Patients diagnosed with breast cancer had low levels of Mammastatin inserum at the time of diagnosis as compared with levels in normal patientserum. Mammastatin levels generally rose on the hormonal cycling,adjuvant chemotherapy protocol. Levels of Mammastatin fluctuated on thisprotocol. Mammastatin levels were undetectable in patients with advanceddisease, before death. The patient data sorted into four groups, asshown in the table below.

I. Group of patients whose serum Mammastatin levels continued to raiseduring therapy.

II. Group of patients whose serum Mammastatin levels increased initiallyduring therapy, but then became undetectable.

III. Group of patients whose serum Mammastatin levels rose duringtherapy, but then fluctuated widely.

IV. Group of patients who had low serum Mammastatin levels which becameundetectable with therapy.

Summary of Mammastatin Levels in Patient Sera Group Number Days FollowedOutcome I.  4 p 280 +/− 100 Remission II. 14 p 500 +/− 220 Deceased III.10 p 380 +/− 280 Variable IV.  5 p 290 +/− 150 Deceased

EXAMPLE 16 In vivo Efficacy of Mammastatin

CD-1 Nu/Nu homozygate, female, six week old mice (Charles River) weresupplemented with Estrogen via slow release pellets, 0.72 mg/pellet, 60day release of 17-beta estradiol (Innovative Research #SE-121). Estrogensupplemented mice were injected with 3×10⁶ MCF-7 cells 100 μl perinjection in 60% matrigel. Two injections were administered, one perflank. After seven days of tumor cell growth, Mammastatin wasadministered. Test mice received 1, 2, or 5 μg of Mammastatin inproduction media at 2 day intervals for a period of six weeks. Controlmice were injected with BSA, or were not injected with tumor, but withthe inhibitor alone.

Tumor size was measured at the point of greatest diameter at weeklyintervals and averaged for treatment group. The results are shown inFIGS. 13A-13C, with tumor size plotted as the mean diameter±standarddeviation.

This animal study was repeated using MDA-231 tumor cells. Cells wereinjected at a concentration of 2×10⁶ cells per injection as describedabove for MCF-7 cells.

The results are shown in FIGS. 14A-14C.

The results shown were not as great as expected. The animals wereinjected by tail vein, resulting in less than the needed blood dose.Subsequent studies using intraperitoneal injection have resulted in moreeffective treatment. At doses of 5 ug/mouse and higher, tumor growth isabrogated.

EXAMPLE 17 Retrovirus Expression of Mammastatin

The Mammastatin cDNA (2.4 kilobase (kb) insert) was subcloned into aretroviral expression vector. The vector was used to transfect 3T3fibroblast cells. Transfected cells were harvested, lysed, and the celllysate analyzed by Western Blot.

As shown in FIG. 12, 3T3 cells transfected with the Mammastatin-carryingretrovirus, expressed phosphorylated Mammastatin.

EXAMPLE 18 Expression of Mammastatin in Baclovirus and Cos 7 Cells

FIG. 16 shows the production of recombinant Mammastatin in Cos-7 MonkeyKidney Cells. Cell lysates were probed with the 7G6, anti-mammastatinmonoclonal antibody. Lane A of FIG. 16 is 25 μg NHMC-20, normal humanmammary cell lysate (positive control); lane B is 25 μg Cos-7 celllysates, transfected with pcDNA3 (negative control); lane C is 10 μgCos-pMammB cell lysate, transfected with pcDNA3/mammastatin construct;and lane D is 20 μg Cos-pMammB cell lysate. Experiment was repeated 3times with similar results.

Induction of recombinant mammastatin expression in Cos-7 cellsdemonstrates that the mammastatin gene codes for authentic mammastatin.Furthermore, the observation that Cos-7 cells express the differentforms of mammastatin associated with phosphorylation of the proteinsuggests that mammastatin will be phosphorylated and active whenproduced in eucaryotic cell lines other than human mammary cells. Stabletransfectants have been selected to allow perpetual synthesis ofrecombinant mammastatin.

EXAMPLE 19 Production of Mammastatin by Normal Human Breast EpithelialCells in Culture

Healthy breast tissue was obtained from reduction mammoplasty, sterile,and direct from the operating room. The tissue was minced under sterileconditions in a laminar flow hood in a solution containing 4 units pergram of type III collagenase (Life Sciences, Bethesda, Md.). The mincedtissue was incubated overnight in a shaking water bath at 37° C. toallow collagenase digestion.

Collagenase digested breast tissue, a viscous fluid containing a varietyof cell types and lipid released from adipose cells, was centrifuged toseparate lipid, aqueous solution, and other cell types. Thecollagenase-digested material was spun at 1000 rpm in a table topcentrifuge at room temperature for 5 minutes. Adipose cells and freelipid partitioned to the top half of the centrifuge tube, and werewithdrawn by aspiration and discarded. The aqueous supernatantpositioned above the cell pellet was also withdrawn by aspiration anddiscarded. The remaining cell pellet was washed with sterile solutionsof mammalian growth media, DMEM, pH 7.4. The washing was continued untilthe supernatant from the washes was no longer turbid (for example about4 washes). The washed cells were resuspended in growth media and allowedto settle by gravity for 30 minutes at 40° C. Because red blood cellsare enucleated and are less dense than nucleated epithelial cells, thisprocedure resulted in removal of the red blood cells from the sedimentedepithelial cells, by withdrawing the supernatant containing the redblood cells. This sedimentation procedure was repeated until no redcolor remained in the cell pellet, e.g., about 2 times. The remainingcell pellet was resuspended in a nutrient rich DMEM/F12 growth mediacontaining 5% equine serum, 10 μg/ml epidermal growth factor, 100 ng/mlof cholera toxin, 500 ng/ml hydrocortisone, 10 μg/ml insulin, 100units/ml penicillin and streptomycin, and 1 mM concentration of calciumchloride. Physiological concentrations of calcium helped to promote cellattachment and outgrowth in cell culture. The cell suspensions wereincubated in sterile tissue culture flasks at 37° C. with a 5% CO₂concentration.

Initial cultures of normal breast tissue contain a mixed cellpopulation. The adipocytes, neurons, and vascular tissue aresignificantly reduced by the differential centrifugation processdescribed above. Connective tissue cells are present in significantamounts. To remove non-epithelial cells, a differential attachmentmethod was used. Fibroblasts, neurons, and other cell types in breasttissue all attach to tissue culture plastic more rapidly than epithelialcells. In addition, all of these cell types are removed from tissueculture plastic by trypsin more rapidly than epithelial cells. To enrichthe cultures for breast epithelial cells, cultures beginning to form amonolayer (5-7 days after initial plating) are treated with atrypsin:EDTA solution (250:1) molar ratio. The majority of cells wereremoved within 5 minutes of incubation at 37° C. The remaining attachedcells were more than 90% epithelial breast cells. These cells were savedand returned to the growth medium described above with 40 μM calciumchloride. The fibroblast cells were removed from the trypsinized cultureflasks, collected by centrifugation, resuspended in growth medium, andplated onto tissue culture plastic for 30 minutes at 37° C. The attachedcells were predominantly fibroblasts. The cells that did not attach weresignificantly enriched for fibroblast cells (50-80%). These suspendedcells were removed and allowed to settle in fresh tissue culture flasks.This process was repeated twice to obtain cell populations that werepredominantly epithelial. Because cholera toxin promotes epithelial cellgrowth and inhibits fibroblast growth, and because fibroblasts do notgrow well in reduced calcium, the cultures were approximately 100%epithelial within one week in the low calcium medium described above.These cultures of normal human mammary cells (NHMC) produced Mammastatininto the culture medium.

Nutrient medium used to grow the NHMC contains 5% equine serum, which isnot acceptable for human injection. The equine serum proteins musteither be purified away from the mammastatin protein, or the cells grownin medium devoid of the serum. Normal cells can only be maintained inthe absence of serum for about seven to ten days. In order to produce asignificant quantity of serum free mammastatin over a prolonged periodof time, the cells were alternately grown in serum-free andserum-containing medium.

NHMC were grown to complete confluence in growth media as describedabove. The cells began to bud in solution as they grew, when cellscovered the available surface of the flask, budding cells were collectedand transferred to new flasks. Confluent flasks were rinsed three timeswith sterile saline, with a five minute saline incubation between washesto remove serum protein. Cells were then provided with serum free“production medium” that was essentially the growth medium devoid ofserum, cholera toxin, and hydrocortisone. Cells were maintained on theproduction medium for about 4 days (96 hours), with collection of themedium, and a return of the cells to growth medium for at least fourdays. The typical batch size for Mammastatin produced in this way was1-2 liters. Mamastatin has also been produced in a Bioreactor, theBioflow 3000, New Brunswick Scientific. In this perfusion reactor, cellswere attached on tissue culture treated fibracell disks. Thecell-attached disks were maintained in a basket in the reaction vesseland perfused with media. When NHMC were introduced to the reactor, theypopulate the fibracell disks and were fed by the perfusion of media.Conditioned media was harvested from the reactor and refrigerated.

EXAMPLE 20 Dot Blot Serum Assay for Mammastatin

Serum from a 25 year old healthy female was obtained and compared withserum from a breast cancer patient (Stage IV), an undiagnosed sibling ofthe patient, and from the patient's mother, whose family has a historyof breast cancer. The serum samples were compared in an immunoassay forthe presence of Mammastatin. Blood samples from multiple breast cancerpatients taken on day of diagnosis were also analized in theimmunoassay. Normal human mammary cell (NHMC) conditioned media was usedas a standard control. Standard NHMC mammastatin contained approximately50 ng/ml as determined in a dot blot assay with mammastatin proteinstandard chromatographically purified.

Individual blood samples were collected into vacutainer tubes, and theserum separated from whole blood. Serum samples (250 or 500 μl volume)were applied without dilution to nitrocellulose by suction using a 96well, S&S Dot-Blot manifold. Conditioned medium was prepared asdescribed for Example 19. Samples on the nitrocellulose filters werewashed with Triton X-100 in TBS, blocked with non-fat dry milk (5% inTBS) and incubated with 1 μg/ml of first anti-mammastatin antibody (7G6mouse IgM) in 5% non-fat dry milk for 1.5 hours at room temperature,followed by incubation with 1 μg/ml of second antibody (goat anti-mouseIgM conjugated to alkaline phosphatase) in 5% non-fat dry milk for onehour and room temperature. The alkaline phosphatase color reaction wasdeveloped using nitroblue-tetrazolium and BCIP.

As shown in FIG. 15, the amount of Mammastatin in the sample wasquantitated against the standard curve obtained from normal breast cellconditioned medium. Serum obtained from healthy females containedreadily detectable amounts of Mammastatin, as indicated by darklycolored blots, whereas serum from diagnosed breast cancer patients, andfrom undiagnosed family members showed little or no Mammastatin.Additional samples were obtained from breast cancer patients on day ofdiagnosis, from healthy members of a breast cancer patient's family, andfrom healthy females and males. The serum was processed as describedabove in order to analyze Mammastatin. The dot blots were evaluated as“negative or low” or “positive or high” to indicate the intensity of thedeveloped color reaction. Data are shown in the following table.

Sample Number Negative or Low Positive or High Breast Cancer patient 8983 (93%) 6 (7%)  Healthy female 11  2 (18%) 9 (82%) Healthy member ofhigh 4  4 (100%) 0 risk family Male 3 2 1

EXAMPLE 21 Treatment of Human Breast Cancer Patients

Twenty-nine (29) Stage IV breast cancer patients with recurrent breastcancer, who had failed, or were failing on chemotherapeutic regimes weregiven access to Mammastatin protein. The protein was produced asdescribed above for Example 19, and provided in production medium, withthe required dose in a 3 ml injection volume. Patients administered theprotein intravenously according to their prescribed regimen. In general,one daily dose was injected. The selected dose was that which providedphysiological amounts of Mammastatin in the patient's bloodstream, e.g.,5-50 ng/ml in healthy women. The dosage and frequency for each patientare indicated in the table below.

Patient Dose Result Number (ug) Schedule Result 1. 125 daily completeremission followed by relapse* 2. 125 daily complete remission; pain iftherapy stopped 3. 75 daily non-responder* 4. 75 daily partialremission; possible immune reaction** 5. 75 daily non-responder* 6. 125daily partial remission 7. 125 daily partial remission* 8. 75 dailynon-responder* 9. 125 every complete remission third day 10. 125 dailynon-responder 11. 125 daily partial remission 12. 125 dailynon-responder# 13. 150 daily non-responder* 14. 75 daily non-responder#15. 125 daily partial remission 16. 125 daily partial remission 17. 75daily non-responder, infection** 18. 125 daily partial remission 19. 125daily partial remission 20. 125 daily partial remission 21. 125 everynon-responder**; other day alternative therapy 22. 125 every partialremission other day 23. 125 every non-responder other day 24. 125 dailynon-responder 25. 125 daily non-responder 26. 125 daily partialremission 27. 125 daily partial remission 28. 125 every partialremission other day 29. 125 daily % without % without % liver lung orliver Total Responders Responders involvement involvement 29 17 59 81 89*patient deceased **Patient withdrawn from therapy #evidence ofimprovement in jaundice prior to death

Of the group of 29 patients, six, having late stage liver disease, didnot survive. These six patients displayed clinical evidence of liverfailure before receiving Mammastatin, and were not helped by thetreatments. One patient showed signs of decreased jaundice before herliver failed, but all six of these patients appeared to die of nitrogentoxicity common to patients with advanced liver cancer.

Of the remaining patients, two have died of their disease. One appearedto be disease-free after two months of Mammastatin therapy. This patientwas removed from therapy, and relapsed within two months. The patient'sdisease was never brought back under control and she died of liverinvolvement. The second patient died after 4 months of treatment, havingnever shown any sign of response to the therapy. There was no sign oftoxicity in any of these patients, although the dose of Mammastatin inthese latter two patients was increased ten fold.

Of the 19 patients currently receiving Mammastatin therapy, the majorityshow signs of positive benefit and no signs of adverse reaction. It isunclear if three of these patients are receiving any benefit fromMammastatin. The other 16 patients show definite clinical signs ofbenefit including decreased tumor markers (CA15-3 and CA27-29) to normallevels, decreased size of palpable tumor masses, decreased disease asevidenced on MRI scan, and decreased pain. Several of these patientsshow improvement to the point of being considered disease free. However,it has consistently been observed that denying these patients proteinfor periods of three to five days results in resumption of diseaseactivity as evidenced by increased pain, even in patients that show nosigns of disease. Resumption of protein treatments decreases oreliminates the symptoms of increased pain within 2-4 hours.

It has also been observed that Mammastatin levels in the blood declineafter long term treatment, suggesting a negative feedback system. Thisdecline in constant blood levels is successfully avoided by providingMammastatin daily for a period of about 28 days, followed by 2-3 dayswithout protein.

EXAMPLE 22 Recombinant Mammastatin for Human Therapy

Recombinant Mammastatin has been produced in Cos-7 monkey kidney cells,chinese hamster ovary (CHO) cells, and Sf9 insect cells by transfectingthe cells with a plasmid containing the Mammastatin cDNA sequence. TheMammastatin cDNA has been stably integrated into the genomes of theseproducing cell lines, and secrete protein immunoreactive with growthinhibitory activity.

To produce Mammastatin from these cells and isolate the protein forhuman use, the cell lines are grown in serum free medium forapproximately 48 to 72 hours. The media is withdrawn and proteinpurified from conditioned medium, either by ion exchange chromatographyin Tris buffer, pH 7.5, using a sodium chloride gradient from about 0.1M to about 0.5 M, collecting the Mammastatin fraction at about 0.2 M.The protein fraction is then dialyzed against normal saline, diluted ifnecessary, and filter sterilized.

In an alternative method, Mammastatin is produced as a fusion protein inCos7 or SF9 cells. The fusion protein contains a histidine tag (sixhistidine residues) and a Factor X proteinase cleavage site. TheMammastatin expressing cells are cultured, preferably in 1%serum-containing media, the conditioned media is collected and passedover a nickel chelating resin. The His-fusion protein adheres to thecolumn, is washed with 50 mM TRIS, pH 7.5, 0.1 M NaCl, and is slowlyeluted with TRIS-NaCl containing 10 Unit/ml Factor X proteinase. Thisliberates Mammastatin from the His fusion. Mammastatin is separated bymolecular sieve chromatography, or by ion exchange chromatography asdescribed above.

The above specification, examples and data provide a completedescription of the manufacture and use of the composition of theinvention. Since many embodiments of the invention can be made withoutdeparting from the spirit and scope of the invention, the inventionresides in the claims hereinafter appended.

TABLE 1 (1) TGGGGCTCCACCCCGGTGGCGGCCGCTCTAGAACTAGTGGATCCCCCGGG (51)CTGCAGGAATTCGGCACGAGCACGGTGAAGAGACATGAGAGGTGTAGAAT (101)CCGTGGGAGGCCCCCGGCGCCCCCCCGGTGTCCCCGCGAGGGGCCCGGGG (151)CGGGGTCCGCCGGCCCTGCGGGCCGCCGGTGAAATACCACTACTCTTATC (201)GTTTTTTCACTGACCCGGTCGAGCGGGGGGGCGAGCCCCGAGGGGCTCTC (251)GCTTCTGGCGCCAAGCGCCCGGCCGCGCGCCGGCCGGGCGCGACCCGCTC (301)CGGGGACAGTGCCAGGTGGGGAGTTTGACTGGGGCGGTACACCTGTCAAA (351)CGGTAACGCAGGTGTCCTAAGGCGAGCTCAGGGAGGACAGAAACCTCCCG (401)TGGAGCAGAAGGGCAAAAGCTCGCTTGATCTTGATTTTCAGTACGAATAC (451)AGACCGTGTAAGCGGGGCCTCACGATCCTTCTGACCTTTTGGGTTTTAAG (501)CAGGAGGTGTCAGAAAAGTTACCACAGGGATAACTGGCTTGTGGCGGCCA (551)AGCGTTCATTAGGACGTCGCTTTTTGATCCTTCGATGTCGGCTCTTCCTA (601)TCATTGTGTAGCAGAATTCACCAAGCGTTGGATTGTTCACCCACTAATAG (651)GGAACGTGAGCTGGGTTTAGACCGTCGTGAGACAGGTTATTTTTACCCTA (701)CTGATGATTGTTTGTTGCCATGGTTATCCTGCTCAGTACGAGAGGAACCG (751)CAGGTTCAGACATTTGGTGTATGTGCTTGGCTGAGGAGCCAATGGGGCGA (801)AGCTACCATCTGTGGGATTATGACTGACGCTCTAAGTCATGAATCCCGCC (851)CAGGCGGAACGATACGGCAGCGCCGCGGAGCCTCGGTTGGCCTCGGATTA (901)GCCGGTCCCCCGCCTGTCCCCGCCGGCGGGCCGCCCCCCCCCCTCCACGC (951)GCCCCGCGCGCGCGGGAGGGCGCGTGCCCCGCCGCGCGCCGGGACCGGGG (1001)TCCGGTGCGGAGTGCCCTTCGTCCTGGGAAACGGGGCGCGGCCGGAAAGG (1051)CGGCCGCCCCCTCGCCCGTCACGCACCGCACGTTCGTGCTCGTGCCGAAT (1101)TCGGCACGAGTGCACCCATTCACAATATACATACAAGTGCATGTATCTTT (1151)ATGATATAATGAATTCTTTTCCTTTGGGTAGATATCCAGTAGTGGGATTG (1201)CTAGATCACCTGGTAGTTCTATTTCTGGTTTATTTAGAAATCTTCATACT (1251)GATTTCCATAGAGGTTGTACAAATTTACATCCCTACCAAAGTGATTTTTT (1301)TAAATATGAAAGAATGGTCTGGAGAAATGCCCCTCATTAGTATCCCCCTT (1351)TTACCTCTCTACTGCAGAATGACTTCAAGGGGTACAGGTATTTACAAGTT (1401)TCATTATACAGACAAATTGAATATTGAAATTTTCTGCATAAGAGGCACAG (1451)ATTTTAGGATTCAAAGTTGTATGAACAAGGACAAGTGCTCTAGGGACTTG (1501)CAAAGCTGGAATTGGAAATCTCAGATGAAATACATTTCTAGTAGTACCAC (1551)CAGCATATATTCTACTGAATTGGCTTTTGTGATCATCATTAATACCTACT (1601)TATTAAAACTAATGAAAAGGGTTTATATCAAATATACTTTAAGGTATAAA (1651)AATCAAATTATAGGTAAAGCTGTTTTCTTTAGCATTTTAATTTCAAAACA (1701)TAAAATAGCTACCGTCTATTGGGCATTTATACTGTACGAGACACTGTGTT (1751)TGTCACATTTCAAAAATGTTCTCATGGTAATGTTCACAATAATTCTGTCG (1801)GGTGAGAAAATAGTCTTACCGTAGTAAGACTATTCAGTAAAACGAAACCT (1851)CTGAACCTTGGAGTTCAACTTGCGCAAAGTTAGTAACAGGACTAGGACTT (1901)GAACCTGAACCATCACACTCGAGATCTCTCCATACCACACTGCTAGCACA (1951)TGTGCCTGTCATCTTATTCCTGGCTCCCTTTTTTATTTCCTTTCCCTTCC (2001)TCCCACAACCCCTTTTTCCCCCCATTTCTTTCTTTCTTTTTATTTGTTAA (2051)TTACATAACTAATACATGTTTATGAGAACAATTGATATAGCACAAAAGGA (2101)TATAAAGTACGGGGGAGTGATAGCTCATCCCTGTAATCCTAGCACTTTGG (2151)AAGGCCAAGGCAGGCAGATCACTTTGAGTCCAGAGTTCGAGACCAGCCTG (2201)GGCAACATGGTGAAA-CCCTGTCTCTACAAAAAAATACAAAAAATTTAGC (2250)CGGGCGTGCTGGCACAGACCTGTAGTCTCAGCTACTCTGAGGGCTGAGGT (2300)GGGAAGATTGATTGAGCCCAGGAGGTGGAAGCTGCAGCAGTGCGCTGAGA (2350)TTGCGCCATTGCACTCCAGCCTGGGTGAGAGAGAGAGACCCTGTCTCCAA (2400)AAAAAAAAAAAAAAAAAAA

1. A method comprising: administering to a patient diagnosed as havingbreast cancer a continuously repeating regimen comprising administeringphosphorylated mammastatin-B for 25-28 consecutive days followed by 2-3days without protein administration, wherein said administration ofphosphorylated mammastatin-B provides a therapeutic blood concentrationof greater than 50 ng/ml and wherein said administration is performed totreat breast cancer.
 2. The method of claim 1, where, in the step ofadministering, the phosphorylated mammastatin-B protein has anapproximate molecular weight of about 53 kDa or about 49 kDa.
 3. Themethod of claim 1, wherein said dose is about 75 μg/day to about 150μg/day.
 4. The method of claim 3, wherein said dose is about 125 μg/day.5. The method of claim 1, wherein said administering is repeated tomaintain a therapeutic blood concentration of said phosphorylatedmammastatin-B protein in the patient.
 6. The method of claim 1, whereinsaid administering provides a therapeutic blood concentration of about500 ng/ml.
 7. The method of claim 1, wherein said administering is byinjection, infusion, or slow-release depot.
 8. The method of claim 1,wherein said administering comprises administering the phosphorylatedmammastatin-B protein once a day.
 9. The method of claim 1, wherein saidadministering comprises administering the phosphorylated mammastatin-Bprotein by continuous infusion.
 10. The method of claim 1, wherein thephosphorylated mammastatin-B protein is administered as apharmaceutically acceptable composition.
 11. The method of claim 1,wherein the said phosphorylated mammastatin-B protein is recombinantlyproduced.
 12. The method of claim 1, wherein said phosphorylatedmammastatin-B protein is produced by culture of normal human mammarycells.